This invention relates to the field of immobilized biologicals for use in diagnostics or other analytical testing. More specifically, it has been found that the application of certain polymeric coatings onto microporous support matrices provides two advantages of major significance to this art. First, it simplifies the procedures required for immobilizing the biological onto the membrane. Second, it essentially eliminates nonspecific binding of protein ont the support surface. Diagnostic assays typically utilize a microporous matrix as a support for a bioaffinity agent, which detects the presence of a particular desired protein (the "target" protein). These bioaffinity agents, usually antibodies to the target protein, are immobilized in some manner on the matrix to yield an "assay matrix" which is used in the diagnostic testing.
Diagnostic membranes are conventionally prepared from microporous membranes, such as nylon, in the following manner. A solution containing the bioaffinity agent is dotted onto the membrane and dried, becoming immobilized on the membrane by passive adsorption. Next, a blocking step is required for the prevention of nonspecific binding of either the target protein or a recognition protein or agent to the target protein. Nonspecific binding across the entire membrane renders the assay inaccurate and unreadible. The blocking step conventionally comprises thoroughly coating the membrane with a protein in order to fill, or block, the nonspecific binding sites available on the support surface.
The assay typically is conducted by passing test fluid through the membrane. If the target protein is present in the test fluid, it will bind to a unique binding site of the primary antibody immobilized on the membrane. The membrane then is treated with a recognition conjugate, consisting of a recognition antibody (which binds to a second binding site on the target protein) coupled to an enzyme, such as horseradish peroxidase, which can be induced to generate a color change under positive test conditions. The membrane is rinsed and treated with a substrate for the enzyme-mediated color change reaction. If the target protein was present in the test fluid, it will have become bound to the primary antibody and, in turn, will have bound the recognition conjugate. The enzyme portion of the recognition conjugate will react with the substrate, producing an easily detected color change for a positive assay. If the protein was not present, the recognition conjugate will not be bound to the membranes and no color change will occur upon treatment with the substrate.
It can be seen that the occurance of nonspecific binding of proteins will seriously impair the usefulness of diagnostic assays of this type. Untreated microporous membranes or particulate support matrices display a strong propensity to adsorb proteins on contact. Thus, any protein present in the test fluid would be adsorbed over the whole support, rather than just being bound to the primary antibody. It is preferred to have an assay system in which a positive result is readily and clearly detectable as a sharp colored dot or pattern on a contrasting background.
Even more detrimental to this assay technique is nonspecific adsorption of the recognition conjugate. The utility of the assay depends upon the recognition conjugate binding only to the target protein, which has been bound by the immobilized primary antibody. If the recognition conjugate can be adsorbed directly onto the support itself, false positives or blinding of the results by a color change over the whole support surface will occur, rendering the assay useless.